DNA polymerases from the red sea brine pool

ABSTRACT

A DNA polymerase composition for amplifying nucleic acids can be tolerant of extreme conditions.

This application is a continuation of prior U.S. application Ser. No.15/302,986 filed Oct. 8, 2016, which is a national phase applicationunder 35 U.S.C. § 371 of International Application No. PCT/IB2015/001423filed Apr. 10, 2015, which claims priority to U.S. ProvisionalApplication No. 61/978,406, filed Apr. 11, 2014, which are herebyincorporated herein by reference in their entirety.

REFERENCE TO SEQUENCE LISTING

-   -   The Sequence Listing submitted Oct. 3, 2019, as a text file        named “18605.0094PCT_SL.txt,” created on Oct. 14, 2015, and        having a size of 53,211 bytes is hereby incorporated by        reference.

FIELD OF THE INVENTION

The present invention relates to DNA polymerases.

BACKGROUND

Polymerase chain reaction (PCR) is a method for the rapid andexponential amplification of target nucleic acid sequences. It has foundnumerous applications in gene characterization and molecular cloningtechnologies including the direct sequencing of PCR amplified DNA, thedetermination of allelic variation, and the detection of infectious andgenetic disease disorders. Various thermostable DNA polymerases havebeen used for PCR applications; for example, Taq polymerase isolatedfrom Thermus aquaticus (Taq), pfu polymerase derived from Pyrococcusfuriosus, KOD polymerase isolated from Thermococcus kodakaraensis, andVent™ DNA polymerase isolated from Thermococcus litoralis (Tli). See,for example, U.S. Pat. Nos. 6,008,025, 5,545,552 and 5,489,523, each ofwhich is incorporated by reference in its entirety.

SUMMARY

In one aspect, a DNA polymerase composition for amplifying nucleic acidsincludes an isolated DNA polymerase having an amino acid residue havingat least 80% homology with the sequence of SEQ ID NOS:1-4. Thepolymerase can be isolated from a brine pool thermophilic archaeaspecies. The polymerase can have about 40% sequence homology to DNApolymerase isolated from Thermococcus litoralis.

In certain embodiments, the polymerase retains at least 100% of itsoptimal DNA polymerase activity in the presence of chloride ion at aconcentration as high as 300 mM. The activity increased with increasingthe chloride ion concentration with 300 mM being the optimalconcentration.

In certain embodiments, the polymerase retains at least 50% of itsoptimal DNA polymerase activity in the presence of sulfate ion at aconcentration as high as 300 mM. The activity increases with increasingthe sulfate ion concentration with 100 mM being the optimalconcentration and decrease to 50% at sulfate concentration of 300 mM.

In certain embodiments, the polymerase retains 100% of its optimal DNApolymerase activity in the presence of K-glutamate at a concentration ashigh as 250 mM. The activity increases with increasing the K-glutamateconcentration with 250 mM being the optimal concentration.

In certain embodiments, the polymerase retains at least 50% of itsoptimal DNA polymerase activity in the presence of Zn²⁺ ion at aconcentration as high as 1 mM. The activity is optimal at 0.5 mM Zn²⁺ion concentration and decrease to 50% at 1 mM.

In certain embodiments, the polymerase retains 100% of its DNApolymerase activity in the presence of Mg²⁺ ion at a concentration ashigh as 100 mM. The activity increases with increasing the Mg²⁺ ionconcentration with 100 mM being the optimal concentration.

In certain embodiments, the polymerase has a DNA extension rate ofgreater than 450 bases per second.

In certain embodiments, the polymerase has a DNA extension processivityof an average of 2000 bases per one cycle of DNA binding event.

In certain embodiments, the polymerase has a DNA proofreading activitythat is at least 2 fold more active than pfu polymerase.

In certain embodiments, the polymerase retains its stability 100% afterbeing heated at 65° C. for 15 minutes.

In certain embodiments, the polymerase is active at room temperature.

In certain embodiments, the polymerase retains 100% of its DNApolymerase activity in pH conditions between 7.5-9.0.

In another aspect, a method for amplifying nucleic acid can includereacting DNA as a template, a primer, dNTP and the DNA polymerasecomposition, and extending the primer to synthesize a DNA primerextension product.

In other aspect, a method for amplifying nucleic acid where the abilityof the polymerase to extend DNA under high salt and metal ionconcentration and different type of metal ions, enables its utilizationto improve currently available molecular biology, biochemical andbiophysical techniques.

In another aspect, a kit for amplifying nucleic acid can include the DNApolymerase composition.

In another aspect, vector can include a gene encoding the DNApolymerase.

In another aspect, a plasmid can include a gene encoding for arecombinant form of the DNA polymerase.

Other aspects, embodiments, and features will be apparent from thefollowing description, the drawings, and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is an image comparing BR3 polymerase and pfu polymeraseactivities at different salt concentrations. Salt used in theseexperiments is NaCl. BR3 and pfu concentrations are 50 nM. FIG. 1B is animage of KOD polymerase activities at different salt concentrations.Salt used in this experiment is KCl and KOD concentration is 50 nM.

FIGS. 2A-2C are images comparing BR3 polymerase and pfu polymeraseactivities in the presence of different salts. Salts used in theseexperiments are: KCl (2A), NH₄(SO)₄ (B) and K-Glutamate (C). BR3 and pfuconcentrations are 50 nM.

FIGS. 3A-3C are images comparing BR3 polymerase and pfu polymeraseactivities in the presence of different metal ions. Concentrations ofmetal ions used in (A) and (B) is 1 mM for MgCl2, MnCl₂, CaCl₂, ZnSO₄,LiCl. BR3 and pfu concentrations are 50 nM.

FIG. 4A is an image comparing BR3 polymerase and pfu polymeraseactivities in the presence of different Mg²⁺ concentrations. BR3 and pfuconcentrations are 50 nM. FIG. 4B is an image of KOD polymeraseactivities in the presence of different Mg2⁺ concentrations.

FIG. 5 is an image comparing BR3 polymerase and pfu polymeraseproofreading activities.

FIG. 6 is an image and a graph depicting the single molecule assay usedto measure rate and processivity of BR3 and pfu polymerases and thehistograms of rate and processivity of DNA synthesis by BR3 and pfupolymerases.

FIG. 7 is an image depicting the thermal stability of BR3 polymerase.

FIG. 8 is an image depicting the ddNTP incorporation efficiency of BR3polymerase and the strategy to engineer its active site to incorporateddNTP.

FIG. 9 represents the sequences of BR1, BR2, BR3, and BR6 polymerases.

FIG. 10 represents the open reading frame sequence of the BR3 polymerase(SEQ ID NO:3).

FIG. 11 represents primary sequence alignment of BR3, KOD, Pfu and Tlipolymerases. Sequences are color-highlighted based on common domainstructures in DNA polymerases, key amino acids involved in catalysis arein bold red and blue and cysteine residues involved in thermal stabilityof the polymerase structure are in bold yellow.

DETAILED DESCRIPTION

The deep-sea anoxic brines of the Red Sea are considered to be one ofthe most remote, challenging and extreme environments on Earth, whileremaining one of the least studied. Approximately 25 such brine-filledpools are currently known, with all of them being anoxic, highly salinedeep-sea water bodies with elevated temperatures, heavy metalconcentration with different types of metal ions that formcharacteristically sharp gradient-rich interfaces with the overlayingsea water. See Backer H & Schoell M (1972) New deeps with brines andmetalliferous sediments in Red Sea. Nature-Physical Science240(103):153, and Hartmann M, Scholten J C, Stoffers P, & Wehner F(1998) Hydrographic structure of brine-filled deeps in the Red Sea—newresults from the Shaban, Kebrit, Atlantis II, and Discovery Deep. MarGeol 144(4):311-330, each of which is incorporated by reference in itsentirety. In contrast to frequent geological and geochemical studies,very few studies have focused on the microbiology of the deep-sea brinesof the Red Sea, and none have concentrated on their biotechnologicalapplications. Initial cultivation-independent and cultivation-basedstudies have provided a first glimpse on the unexpected enormousbiodiversity of the local microbial communities, with identification ofseveral new groups and the isolation of new extremophilic microorganismsthat thrive in these environments. See Antunes A, Eder W, Fareleira P,Santos H, & Huber R (2003) Salinisphaera shabanensis gen. nov., sp nov.,a novel, moderately halophilic bacterium from the brine-seawaterinterface of the Shaban Deep, Red Sea. Extremophiles 7(1):29-34, AntunesA, et al. (2008) A new lineage of halophilic, wall-less, contractilebacteria from a brine-filled deep of the Red Sea. J Bacteriol190(10):3580-3587, Antunes A, et al. (2008) Halorhabdus teammate spnov., a non-pigmented, extremely halophilic archaeon from a deep-sea,hypersaline anoxic basin of the Red Sea, and emended description of thegenus Halorhabdus. Int J Syst Evol Micr 58:215-220, Eder W, Ludwig W, &Huber R (1999) Novel 16S rRNA gene sequences retrieved from highlysaline brine sediments of Kebrit Deep, Red Sea. Arch Microbiol172(4):213-218, Eder W, Jahnke L L, Schmidt M, & Huber R (2001)Microbial diversity of the brine-seawater interface of the Kebrit Deep,Red Sea, studied via 16S rRNA gene sequences and cultivation methods.Appl Environ Microb 67(7):3077-3085, and Eder W, Schmidt M, Koch M,Garbe-Schonberg D, & Huber R (2002) Prokaryotic phylogenetic diversityand corresponding geochemical data of the brine-seawater interface ofthe Shaban Deep, Red Sea. Environ Microbiol 4(11):758-763, each of whichis incorporated by reference in its entirety. Because of the unusuallyharsh conditions of this environment, it is highly likely that theresiding microbes developed novel metabolic pathways, transport systemsacross their membranes, enzymes, and chemicals in order to survive.

This environment presents the harshest conditions for the DNAreplication machinery as well as DNA processing enzymes to copy and tomaintain the genomic DNA, indicating the utilization of novel adaptivemechanisms and nucleic acid binding proteins. Archaeal and bacterialspecies from the brine pool can be used to screen for novel DNAsequencing polymerase and other key DNA modifying enzymes.

The classical chain-termination method used for DNA sequencing, theSanger method, relies on using a DNA polymerase that has high rate ofincorporation of the chain terminator dideoxynucleoside triphosphate(ddNTP). See Tabor S & Richardson C C (1995) A single residue in DNApolymerases of the Escherichia coli DNA polymerase I family is criticalfor distinguishing between deoxy- and dideoxyribonucleotides. Proc NatlAcad Sci USA 92(14):6339-6343, which is incorporated by reference in itsentirety. DNA polymerases normally catalyze a nucleophilic attack of the3′-OH group of the primer on the α-phosphate of the incoming dNTP. TwoMg²⁺ ions are required in this reaction to align the primer/templatestrand and the incoming dNTP and to mediate the substitutionnucleophilic attack reaction. See Johnson A & O'Donnell M (2005)Cellular DNA replicases: components and dynamics at the replicationfork. Annu Rev Biochem 74:283-315, and Hamdan S M & Richardson C C(2009) Motors, switches, and contacts in the replisome, each of which isincorporated by reference in its entirety. The lack of the 3′-OHnuleophilic group in the ddNTP is the reason for its action as a DNApolymerase inhibitor. See Tabor S & Richardson C C (1995) A singleresidue in DNA polymerases of the Escherichia coli DNA polymerase Ifamily is critical for distinguishing between deoxy- anddideoxyribonucleotides. Proc Natl Acad Sci USA 92(14):6339-6343, whichis incorporated by reference in its entirety. During sequencing, DNAsynthesis reaction starts from a specific primer and ends upon theincorporation of ddNTP. By using either dye- or radiolabel-based ddNTP,the identity of these products can be mapped. In general, DNApolymerases polymerize the dNTP with very high accuracy (1 mistake per10³-10⁵ incorporated nucleotides) and encode for a proofreadingexonucleases activity to remove misincorporated nucleotide (accuracyincreased to 1 mistake per 10⁵-10⁷ incorporated nucleotides). Therefore,the ideal DNA sequencing polymerase will have high rate and highprocessivity of DNA synthesis, high accuracy of dNTP incorporation,proofreading exonuclease activity, high thermal stability and high rateof incorporation of ddNTP.

Indeed all these properties have been fulfilled with the introduction offour DNA polymerases to the market that are isolated from archaeaspecies, Pyrococcus furiosus DNA polymerase (pfu DNA Pol), Thermococcuslitoralis Vent™ DNA polymerase (Vent DNA Pol), Thermococcus Kodakarensis(KOD Pol) and Thermus Aquaticus DNA polymerase (Taq Pol) (Table 1).

TABLE 1 Characteristics of DNA Polymerases. The table is adopted fromTakagi M et al. (1997) Characterization of DNA polymerase fromPyrococcus sp. strain KOD1 and its application to PCR. Applied andEnvironmental Microbilogy 63(11): 4504-4510 KOD PFU Taq SpeciesThermococcus Pyrococcus Thermus kodakaraensis furiosus aquaticus YT-1Fidelity 0.0035 0.0039 0.013 Elongation rate 106-138 25 61(bases/second) Processivity >300 <20 unavailable (nucleotide bases)

Disclosed herein is a method of utilizing DNA polymerases from the Brinepool with the aim of generating a commercial polymerase that has therobust reaction features of utilizing wide range of salt and metal ionconcentration and metal ion types as well as enhanced rate processivityand proofreading activity. Also disclosed is a method and a compositionfor amplifying nucleic acid where the ability of the polymerase toextend DNA under high salt and high metal ion concentrations, in thepresence of different type of metal ions, and high temperatureconditions enables its utilization to improve currently availablemolecular biology, biochemical and biophysical techniques. None of theconventional polymerases are ideal for the harsh conditions like highsalt concentrations, high metal concentrations, and high temperature,let alone combinations of two or more of these conditions. Disclosedherein is a polymerase that can not only tolerate under one of theseharsh conditions, but also can tolerate various combinations of thoseconditions, e.g. high salt and metal concentrations, high metalconcentration and high temperate, high salt concentration and hightemperate, or high salt and metal concentrations and high temperate.

Robust DNA sequencing enzymes isolated from the Brine pool can sustainwide range of salt and metal ion concentrations, different type of metalions and wide range of pH during PCR. Four clones of DNA polymerase havebeen identified from the brine pool (FIG. 9, Table 2). These polymerasesfrom microorganisms from the Brine pool can be used for conducting a PCRreaction at wide range of buffer conditions and metal ions concentrationand types. Optimization of PCR remains tricky as it might requirescreening for the appropriate salt and metal ion concentrations thatlead to high yield, high processivity and accuracy of the amplified DNAfragment. The ability of thermal archaea species from the brine pool toreplicate their genome at high salt concentration indicates that theirDNA polymerases binds to the DNA with relatively high affinity, whichcould potentially enhance the sensitivity of the PCR, and couldtherefore tolerate wide range of salt concentrations. Furthermore, theability of these DNA polymerases to tolerate high metal ionconcentrations indicates that they can work at wide range of metal ionconcentrations. Finally, the ability of these DNA polymerases totolerate different type of metal ions indicates that they can work atwide range of metal ion types. DNA polymerases from the brine poolthermophilic archaea species were cloned, expressed, purified andcharacterized.

TABLE 2 Identification of DNA Polymerases from the Brine Pool.Homologous size species Enzyme % homology Clone 1 2100bp, 700aaCandidatus DNA 25% (SEQ ID NO: 1) Nitrososphaera Polymerase B gargensisClone 2 2352bp, 784aa Thermococcus DNA 42% (SEQ ID NO: 2) celerdependent Polymerase Clone 3 2457bp, 819aa Thermococcus DNA 42% (SEQ IDNO: 3) litoralis dependent Polymerase Clone 6 2400bp, 800aa CandidatusDNA 42% (SEQ ID NO: 4) Nanosalinarum dependent Polymerase

Especially, Clone 3 (termed BR3, FIG. 10) which is 42% homologous to DNAdependent polymerase of Thermococcus litoralis shows much more robustproperties than any known commercially available DNA polymerases thatare used in PCR and DNA sequencing.

BR3 tolerates extremely versatile buffer conditions. FIGS. 1A and 1Bshow that BR3 polymerase retains its optimal activity up to 300 mM NaClwhereas pfu and KOD polymerases retains their optimal activity only upto 10 mM. BR3 polymerase also tolerates different types of salts. FIG. 2shows that BR3 polymerase tolerates up to 300 mM KCl (FIG. 2A), up to100 mM (NH₄)₂SO₄ (FIG. 2B), and up to 250 mM K-Glutamate (FIG. 2C). Therange is at least 15-30 fold higher than pfu polymerase.

FIG. 3 shows that BR3 polymerase shows much higher activity than pfupolymerase in the presence of MgCl₂ and MnCl₂ when the saltconcentration is high (FIG. 3B). Moreover, BR3 polymerase shows highmetal ion resistance, for example 0.1-100 mM MgCl₂ (FIGS. 4A and 4B).This range is 10-fold higher than pfu polymerase and KOD polymerase.When the salt concentration is low (FIG. 3A), pfu polymerase showshigher activity in the presence of MgCl₂ and MnCl₂. Neither polymerasesshow a significant activity when calcium or lithium ions are present. Itis notable that in the high salt concentration, BR3 polymerase retainsactivity in the presence of zinc ions (FIGS. 3B and 3C). BR3 polymeraseis the first known polymerase to use zinc ions or any metal ions otherthan Mg²⁺ and Mn²⁺.

BR3 polymerase has at least 2-fold higher proofreading activity than pfupolymerase (FIG. 5). FIG. 5 shows that BR3 polymerase only requires halfthe concentration of pfu polymerase to produce the same activity levelin the presence of up to three mismatches on the primer strand.

FIG. 6 shows the single molecule assay that was used to measure the rateand processivity of BR3 and compare it with pfu polymerase and Table 3shows the results from this measurement, where BR3 polymerase displaysat least 1.5-fold higher rate and processivity than pfu polymerase.

TABLE 3 Comparison of rate and processivity of BR3 polymerase and pfupolymerase Rate Processivity (base/sec) (kb) BR3 polymerase 463.34 ±34.73 2.0 ± 0.3 PFU polymerase  305.5 ± 40.46 1.3 ± 0.1

BR3 polymerase retains the same polymerization activity at pH rangebetween 7.5-9.0. It also showed high thermal stability up to 65° C.(FIG. 7). Its thermal stability can be likely increased by inducing theformation of a highly conserved disulfide bond in the active site ofextreme thermophilic polymerases. BR3 polymerase also discriminates wellagainst the incorporation of ddNTP (FIG. 8). It is highly likely toincrease the incorporation efficiency of ddNTP by mutating F residue inactive site to Y in BR3 (FIG. 8).

These properties make this polymerase ideal to be used in DNA sequencingand molecular biology techniques with minimal reaction optimization andwith different sample types and preparations.

The BR3 polymerase can be produced using recombinant techniques.

As used herein, the terms “polypeptide” and “protein” are usedinterchangeably to refer to a polymer of amino acid residues. The term“recombinant polypeptide” refers to a polypeptide that is produced byrecombinant techniques, wherein generally DNA or RNA encoding theexpressed protein is inserted into a suitable expression vector that isin turn used to transform a host cell to produce the polypeptide.

As used herein, the terms “homolog,” and “homologous” refer to apolynucleotide or a polypeptide comprising a sequence that is at leastabout 50% identical to the corresponding polynucleotide or polypeptidesequence. Preferably homologous polynucleotides or polypeptides havepolynucleotide sequences or amino acid sequences that have at leastabout 80%, at least about 85%, at least about 90%, at least about 91%,at least about 92%, at least about 93%, at least about 94%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,or at least about 99% homology to the corresponding amino acid sequenceor polynucleotide sequence. As used herein the terms sequence “homology”and sequence “identity” are used interchangeably. One of ordinary skillin the art is well aware of methods to determine homology between two ormore sequences, for example, using BLAST.

A mutant or variant polypeptide refers to a polypeptide having an aminoacid sequence that differs from the corresponding wild-type polypeptideby at least one amino acid. In some embodiments, the mutant polypeptidehas about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80,90, 100, or more amino acid substitutions, additions, insertions, ordeletions. For example, the mutant can comprise one or more conservativeamino acid substitutions. As used herein, a “conservative amino acidsubstitution” is one in which the amino acid residue is replaced with anamino acid residue having a similar side chain. Families of amino acidresidues having similar side chains have been defined in the art. Thesefamilies include amino acids with basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine), and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine).

Preferred variants of a polypeptide or fragments a polypeptide retainsome or all of the biological function (e.g., enzymatic activity) of thecorresponding wild-type polypeptide. In some embodiments, the variant orfragment retains at least about 75% (e.g., at least about 80%, at leastabout 90%, or at least about 95%) of the biological function of thecorresponding wild-type polypeptide. In other embodiments, the variantor fragment retains about 100% of the biological function of thecorresponding wild-type polypeptide. In still further embodiments, thevariant or fragment has greater than 100% of the biological function ofthe corresponding wild-type polypeptide. It is understood that thepolypeptides described herein may have additional conservative ornon-essential amino acid substitutions, which do not have a substantialeffect on the polypeptide function.

EXAMPLES

Enzymes: The cDNA fragment corresponding to BR3 (see FIG. 9 SEQ ID NO:3)was amplified by PCR using primers (5′-CACCATGGCAAATCAGACAACAAATGG-3′and 5′-TTATTTGAATTTTCCGAGTTTTACTTGTCG-3′) and cloned into thepENTR-D/TOPO vector (Life Technology). The ORF of BR3 was transferred topDEST17 vector (Life Technology) by using LR Clonase II enzyme mix (LifeTechnology). BR3 is overexpressed in E. coli Rozetta2 (DE3) (Novagen)after transformation with plasmids pDEST17/BR3. The overexpression wasinduced by addition of Isopropyl.beta.-D-1-thiogalactopyranoside (finalconcentration, 1 mM), and cells were harvested after 3 h of incubation.The collected cells were dissolved in Lysis buffer (10 mM Tris-HCl pH8.0, 80 mM KCl, 5 mM 2-Mercaptoethanol, 1 mM EDTA(Ethylenediaminetetraacetic acid)) and incubated on ice with Lysozyme(final concentration, 1 mM) for 30 m, then disrupted by sonication. Thecrude extract was centrifuged to remove cell debris, the supernatant wascollected and ammonium precipitation was performed with 80% saturation.The pellet obtained from ammonium precipitation was dissolved in BufferA (10 mM Tris-HCl pH 8.0, 1 mM EDTA and loaded onto the SephacrylSepharose (GE Healthcare) column. The flow through fraction fromSephacryl Sepharose was collected and diluted enough to reduce EDTAconcentration, then loaded onto HisTrap HP 5 ml (GE Healthcare) and thebound proteins were eluted by Buffer B (10 mM Tris-HCl pH 8.0, 50 mMKCl, 500 mM Imidazole). The peak fractions were collected and passedthrough HiTrap Heparin 1 ml (GE Healthcare) and the fractions containingpure proteins were eluted by making a gradient against Buffer C (10 mMTris-HCl pH 8.0, 50 mM KCl, 1 M KCl). The purified BR3 proteins weredialyzed against Buffer D (50 mM Tris-HCl pH 7.5, 50 mM KCl, 1 mM DTT(dithiothreitol), 0.1% TWEEN® 20 (polysorbate 20), 50% Glycerol). Theprotein concentration was determined by absorbance at 280 nm with theextinction coefficient and molecular weight were calculate based on theamino acid sequence of BR3 protein.

Primer extension and proofreading activity assay: The polymerase andproofreading activities were characterized as published (see LundbergeK. S. et al. (1991) High-fidelity amplification using a thermostable DNApolymerase isolated from Pyrococcus furiosus (Polymerase chain reaction;mutation archaebacteria) frequency; lack; proofreading; 3′40-5exonuclease; recombinant DNA. Gene (108): 1-6 with followingmodifications for the proofreading assay. The 35-mer template containingan internal EcoRI site is annealed to 15-mer Cy3-labeled primers with 0,1, and 3 mismatch nt at the 3′ terminus. Reactions were carried out at45 C.° in 22 μl for 5 min and contained basic buffer (20 mM Tris-HCl pH8.8, 10 mM (NH₄)₂SO₄, 50 mM KCl, 2 mM MgSO₄, 0.1% TritonX-100), 200 μMdNTPs and 1 mM MgCl₂. Of each reaction 10 μl was removed and reactionswere stopped by adding 4 μl of stop solution (100 mM EDTA pH 8.0), theremaining 10 μl of each reaction was digested with 5 u of EcoRI at 37C.° for 30 min. The reactions were terminated by adding 4 μl of stopsolution. The synthesized product was loaded to 15% polyacrylamide/7.5 Murea/1× TBE denaturing gel. The gel was visualized by Typhoon TRIO (GEHealthcare). The polymerization activity of KOD was tested byconventional PCR on primed ssDNA pUC19 plasmid as a template.

Primer extension assay at the single molecule level: DNA synthesis wasmeasured by monitoring the length of individual DNA molecules in realtime as described previously (See Tanner, N. A. et al. (2008)Single-molecule studies of fork dynamics in Escherichia coli DNAreplication. Nature structural & molecular biology (15): 170-176,Jergic, S. et al. (2013) A direct proofreader-clamp interactionstabilizes the Pol III replicase in the polymerization mode. The EMBOjournal (32):1322-1333, and Lee, J. B. et al. (2006) DNA primase acts asa molecular brake in DNA replication. Nature (439):621-624, each ofwhich is incorporated by reference in its entirety. Briefly, ssDNAtemplate containing a biotinylated primer was attached to the surface ofa glass coverslip via one end and to a magnetic bead via the other endin microfluidic flow cell (FIG. 6). The DNA molecules were stretched bya laminar flow that exerted a 2.6 piconewten (pN) drag force on thebeads. Primer extension converts the surface tethered ssDNA (short) todsDNA (long) and increase the length of the DNA as schematicallyillustrated in FIG. 6 and shown in the trajectories in FIG. 6. The assaywas performed at 25° C. in buffer containing (20 mM Tris-HCl pH 8.8, 10mM (NH₄)₂SO₄, 2 mM MgSO₄, 0.1% TritonX-100), 200 μM dNTPs, 1 mM MgCl₂,and either 250 mM KCl in case of BR3 polymerase or 50 mM KCl in case ofpfu polymerase. BR3 and pfu polymerases were used at 50 nM.

Other embodiments are within the scope of the following claims.

What is claimed is:
 1. A composition for amplifying nucleic acidscomprising: an isolated DNA polymerase comprising the sequence of SEQ IDNO:3 and an amount of Ethylenediaminetetraacetic acid (EDTA), Tris-HCland/or dithiothreitol (DTT).
 2. The composition of claim 1, furthercomprising glycerol.
 3. The composition of claim 1, comprising thesequence of SEQ ID NO: 3 and DTT.
 4. The composition of claim 2,comprising an amount of EDTA, Tris-HCl and DTT.
 5. The composition ofclaim 1, comprising the sequence of SEQ II) NO: 3 and an amount of EDTA.6. A composition for amplifying nucleic acids comprising: an isolatedDNA polymerase comprising the sequence of SEQ ID NO:3 and 50% glycerol.